1. Field Of The Invention
This invention relates to a method of forming a stable composite of an enzyme substrate and a solid support. As known in the prior art much of clinical and biochemical separations methodology depends upon the size, shape and other physical characteristics of the material in question. This invention is concerned with the immobilization of carbohydrate enzyme substrates which may be used in affinity chromatography enzyme assay methodology or similar procedures.
2. Description Of The Prior Art.
In the past the importance of conditioning various biological materials for a particular use has been known. Small enzyme substrates have been immobilized (for example see "Assay of Cholinesterase In An Electrode System With An Immobilized Substrate" by Guilbault, G. G. and Iwase, A., Analytica Chimica Acta, V. 85, p. 295-300 (1976)). The particular enzyme substrate discussed herein, starch, has long been recognized as an important commercial product. Many procedures are known for the modification of starches for various functions. For example, U.S. Pat. No. 3,436,309, discloses a one-step process for the conversion of natural or so-called raw starch, to a product comprising a partially hydrolyzed, synthetic resin mixed complex which is used as a sizing for paperboards and the like.
Similarly, many procedures are known to hydrolyze starches, for example, U.S. Pat. No. 3,996,107, discloses a procedure to "liquify" starches under varying conditions with varying enzymes, and U.S. Pat. No. 3,912,590, which are formed by enzymatic hydrolysis at elevated temperatures to form a liquified product.
Also, starches, in granule form have been used to form medical dusting powders and the like. For example, U.S. Pat. No. 2,626,257, shows the use of starch granules for lubricating powders in medical gloves and the like. The patent also discloses that starch is treated with a diether forming halogen compound from either the halohydrin or epoxyhalohydrin groups, for example glycerol dibromo- and dichlorohydrins or epichloro-or epibromohydrin. This procedure produces from the raw ungelatinized starch a partially diether linked granular starch which can withstand harsh sterilization conditions without swelling or breaking. Therefore, the bridging groups are used in the procedure to retard granule swelling and so-called gelatinization. This material was not as flowable as desired and therefore the dry granules were mixed with various flow agents, for example non-hydroscopic metal oxides. Various other procedures for etherifying or esterifying starches are known, for example, U.S. Pat. No. 2,459,108.
Polysaccharides have also been used as support agents to provide for the immobilizing of various other chemical compounds. For example, U.S. Pat. No. 3,914,183, shows the use of a polysaccharide matrix to immobilize proteins containing free amino groups.
No method is believed known which form stable enzyme substrate composites using starch. These immobilized starch composites would find uses in affinity chromatography and in procedures where a precise amount of substrate is needed to perform an enzyme-substrate reaction to quantify the enzyme.